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WO2004078311A1 - Procede de preparation de ligands pour la chromatographie d'interaction hydrophobe - Google Patents

Procede de preparation de ligands pour la chromatographie d'interaction hydrophobe Download PDF

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Publication number
WO2004078311A1
WO2004078311A1 PCT/SE2004/000316 SE2004000316W WO2004078311A1 WO 2004078311 A1 WO2004078311 A1 WO 2004078311A1 SE 2004000316 W SE2004000316 W SE 2004000316W WO 2004078311 A1 WO2004078311 A1 WO 2004078311A1
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Prior art keywords
interaction
ligand
hic
group
ligands
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Inventor
Bo-Lennart Johansson
Jean-Luc Maloisel
Nicolas Thevenin
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Cytiva Sweden AB
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Amersham Bioscience AB
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Application filed by Amersham Bioscience AB filed Critical Amersham Bioscience AB
Priority to JP2006507945A priority Critical patent/JP4570052B2/ja
Priority to CA2517757A priority patent/CA2517757C/fr
Priority to EP04717913A priority patent/EP1599265A1/fr
Priority to US10/547,570 priority patent/US7320755B2/en
Publication of WO2004078311A1 publication Critical patent/WO2004078311A1/fr
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3285—Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B01D15/327—Reversed phase with hydrophobic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers

Definitions

  • the present invention relates to a method of preparing ligands and a medium for separation based on hydrophobic interactions between a target and the ligands, such as hydro- phobic interaction chromatography (HIC).
  • HIC hydro- phobic interaction chromatography
  • the invention also embraces separation media comprising such ligands as well as use thereof to isolate molecules, such as proteins and peptides.
  • chromatography embraces a family of closely related separation methods.
  • the feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile.
  • the sample mixture introduced into the mobile phase, undergoes a series of interactions (partitions) many times before the stationary and mobile phases as it is being carried through the system by the mobile phase.
  • Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual compo- nents under the influence of a mobile phase moving through a column containing the stationary phase.
  • Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
  • HIC hydrophobic interaction chromatography
  • Proteins and peptides usually sequester hydrophobic amino acids in domains away from the surface of the molecule, but still usually have sufficient hydrophobic groups exposed to allow interaction with hydrophobic ligands attached to a stationary phase.
  • An advantage of HIC is that elution conditions are mild, which is preserves biological activity.
  • One kind of available HIC media is based on aromatic interaction mode, such as Phenyl SepharoseTM (Amersham Biosciences, Uppsala, Sweden).
  • aromatic derivatised media can e.g. be prepared by immobilisation on an agarose base matrix of phenyl glycidyl ether under Lewis acid catalysis.
  • HIC media is based on aliphatic interaction mode, such as Butyl SepharoseTM (Amersham Biosciences, Uppsala, Sweden).
  • aliphatic ligands can e.g. as well be prepared in a similar fashion as described above, but this time with the use of aliphatic glycidyl ether.
  • RPC reverse phase chromatography
  • proteins have become of increased interest and consequently the need of efficient methods for purification thereof has increased.
  • ion exchange has been the most prevalent chromatography method for protein purification.
  • proteins are complex molecules that could provide a plurality of possible interaction with a stationary phase.
  • general separation media that utilises both the hydrophobici y of a protein and other interactions simultaneously. Accordingly, there is a need in this field of HIC media that are supplemented with groups that provides one or more additional interaction modes. Consequently, there is also a need of efficient methods for preparing such multi- modal media for use in HIC and RPC.
  • Feist and Danna (“Sulfhydryl cellulose: A New Medium for Chromatography of Mercurated Polynucleotides”. Patricia L. Feist and Kathleen J. Danna, Biochemistry, 20(15), p. 4243-4246) have disclosed a process of preparing sulfhydryl cellulose, which process includes to mix amino ethyl cellulose with an N-acetylhomocysteine thiolactone. The concentration of active sulfhydryl groups is determined by reacting the sulfhydryl cellulose with Ellmans reagent.
  • One object of the present invention is to provide a method of preparing ligands for hydrophobic interaction chromatography, which method is a simple and robust two step synthesis.
  • a specific object is to provide such a method, which allows introduction of multiple functionalities on each ligand.
  • a further object of the invention is to provide a method as mentioned above, which can be immobilised to the desired degree of substitution on a solid support.
  • Figure 1 provides by Scheme 1 an illustration of how a diverse library of multimodal HIC/RPC media can be prepared according to the invention and using homocysteine thiolactone as a scaffold.
  • Figure 2 shows a comparative elution profile of four proteins (myoglobine (1), ribonu- clease A (2), alpha-lactalbumine (3), and alpha-chymotrypsine (4)) on the prior art separation medium Phenyl SepharoseTM 6FF (Amersham Biosciences, Uppsala, Sweden), as described below.
  • Figure 3 shows the elution profile of four proteins (as defined under Figure 2) on Gel 2 according to the invention, as described below.
  • Figure 4 shows the elution profile of four proteins (as defined under Figure 2) on Gel 4b according to the invention, as described below.
  • Figure 5 shows the elution profile of four proteins (as defined under Figure 2) on Gel 7 according to the invention, as described below.
  • Figure 6 shows the elution profile of four proteins (as defined under Figure 2) on Gel 9 according to the invention, as described below.
  • Figure 7 shows a comparative elution profile of four proteins (as defined under Figure 2) on the prior art separation medium Phenyl SepharoseTM HP (Amersham Biosciences, Uppsala, Sweden), as described below.
  • Figure 8 shows the elution profile of four proteins (as defined under Figure 2) on Gel 11 according to the invention, as described below.
  • Figure 9 shows the difference of elution profiles obtained with different multimodal media, and illustrates the influence of the second interactions in RPC mode.
  • spacer is used for a chemical entity that distances a ligand from the base matrix.
  • ligand means herein a chemical entity capable of binding target substances.
  • target substances may be either a compound, which it is desired to isolate or re- move by chromatography, or alternatively an analytical target substance.
  • ion exchange ligands means groups that are charged during the adsorption phase of chromatography.
  • mixed mode ligand and “multimodal ligand”, in the context of this invention, refer to a ligand that is capable of providing at least two different, but co-operative, sites which interact with the substance to be bound. One of these sites gives hydrophobic interaction between the ligand and the substance of interest. The second site typically gives electron acceptor-donor interaction and/or hydrophobic and/or hydrophilic interactions. Electron donor-acceptor interactions include interactions such as hydrogen- bonding, ⁇ - ⁇ , charge transfer, dipole-dipole, induced dipole etc.
  • Electrode donor-acceptor interactions mean that an electronegative atom with a free pair of electrons acts as a donor and bind to an electron-deficient atom that acts as an ac- ceptor for the electron pair of the donor. (See e.g. Karger et al., An Introduction into Separation Science, John Wiley & Sons (1973) page 42.)
  • Typical acceptor atoms/groups are electron deficient atoms or groups, such as metal ions, cyano, nitrogen in nitro etc, and include a hydrogen bound to an electronegative atom such as -OH in hydroxy, and carboxy, -NH- in amides and amines, HS- in thiol etc.
  • the present invention is a method of preparing at least one multimodal ligand for hydrophobic interaction chromatography (HIC), which method comprises the steps of
  • A, B and X irrespective of each other are carbon atoms or any heteroatoms, such as oxygen, sulphur, nitrogen and/or silica, m is any integer between 0 and 4, such as 1-3, preferably 1 or 2, and the functional group N is a nitrogen, which either replaces one X or is attached to any one of A, B and X;
  • ion-exchange ligands also includes groups that under the conditions used in chromatography are transformed into charged species.
  • the ligand prepared ac- cording to the present method does not allow ionic interaction with target compounds via said first and second interactions. It is also understood that said first and second interactions are groups capable of interaction with the same target molecule.
  • the present method is based on the use of three-functional scaffold presenting multiple advantages. It allows the selective introduction in turn of different interactions and the regeneration in the last step at the same time as the introduction of some extra interactions, of a thiol group for further immobilisation on a solid support. Given the high nucleophilicity of the thiol group, the immobilisation of the multimodal ligand via this group insures a homogenous media with a level of substitution that can be easily controlled and analysed. These specific characteristics allow a much shorter, much more efficient and. cost-effective preparation of multimodal HIC media, which should otherwise be prepared via multiple steps of protection and deprotection of the functional groups.
  • A, B, and X are carbon atoms, and m is 1.
  • m can alternatively be an integer above 4, such as 5-500, e.g. 10-250 or more specifically 50-100, depending on the desired size of the ligands.
  • N can be attached to the cyclic struc- ture via a linker, such as conventionally used in separation media, which linker may comprise linear, branched, cyclic saturated, unsaturated and aromatic hydrocarbon groups, e.g. with up to 1-20, such as 1-10 carbon atoms.
  • the scaffold is homocysteine thiolactone.
  • homocysteine thiolactone can be used in an optically pure or racemate form.
  • Homocysteine thiolactone is commercially available, e.g. from Aldrich, catalogue no. HI, 580-2, and CAS no. 6038-19-3.
  • the reagent used in step (b) to provide the primary interaction is defined by the general formula (II)
  • Z is a group which is capable of reacting with the nitrogen of the scaffold; and R is a group that provides the first interaction and can be a linear, branched, cyclic saturated, unsaturated and aromatic hydrocarbon group, which preferably comprises about 1- 20, such as 1-10 carbon atoms.
  • Y represents for example a halogen, such as Br, I, CI, or a mesylate, or a tosylate group
  • the R group provides the first interaction.
  • R provides a hydro- phobic group, such as an aromatic or an aliphatic group.
  • R provides any other group, which is non-charged under the adsorption conditions used, that provides an additional interaction with a target compound, such as electron acceptor-donor interaction, hydrogen bonds etc.
  • R can carry hydroxy groups, halogens, alkoxy and aryloxy and the corresponding thio analogues, and/or amino groups.
  • Carbon chains may at one or more positions be interrupted by heteroatoms for certain applications, ether oxygen, thioether sulphur etc.
  • R may also be carbonyl groups, such as in amides and ketones, and other groups having the comparable stability against hydrolysis.
  • R can contain practically any groups, as long as the final separation medium exhibits a full interval window where it is globally hydrophobic and can function in hydrophobic inter- action chromatography.
  • the present method can use R groups comprised of two or more parts that are functional in binding.
  • R provides two functional groups Li and L 2 that enable primary interaction.
  • step (b) is alkylation, acylation or sulfonyla- tion.
  • step (b) comprises an acylation or a sulfonylation; a combination of alkylation and acylation; or combination of alkylation and sulfonylation.
  • step (c) above which is a ring opening by aminolysis, is performed by adding any suitable amine that carries a secondary interaction, i.e. one or more functionalities capable of interacting with a target compound.
  • a reagent is added, which is comprised of two parts: one amine part and one part that provides one or more functional groups that enable the desired interactions.
  • the secondary interaction can be any one of the ones discussed above in the context of the primary interaction, with the one proviso that at least one of said interactions provides hydrophobic interaction and that none of them are charged groups under the adsorption conditions used.
  • the amine added is defined by the general formula II:
  • L 3 and L 4 comprise functionalities, which can be the same or different.
  • the present invention also comprises a step (d), wherein the thiol of the product obtained from step (c) is reacted with a base matrix comprising a reactive group.
  • the product of this embodiment is a separation medium for HIC, wherein a plurality of multimodal ligands are immobilised to a suitable base matrix.
  • the base matrix of step (d) is commonly in beaded or monolithic form.
  • the base matrix can e.g. be in the form of a gel, preferably a porous gel, or porous beads.
  • the base matrix can e.g. be a membrane, a filter, one or more chips, surfaces, capillaries etc
  • the base matrix of step (d) is made from a natural polymer. Suitable porous polymer beads of natural polymers are either easily performed by the skilled person in this field according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964) or spin- ning disk technique (see e.g.
  • WO 88/07414 Prometic Biosciences Inc) or obtained from commercial sources, such as Amersham Biosciences AB, Uppsala, Sweden.
  • Illustrative tradenames of such useful natural polymer beads are e.g. of the kind known as SepharoseTM or SephadexTM.
  • the base matrix is agarose.
  • ligands are immobilised to a particulate base matrix to a substitution level of about 10-50, such as about 20, ⁇ mol/ml base matrix to provide a HIC medium.
  • the base matrix of step (d) is made from a synthetic polymer, preferably a cross-linked synthetic polymer, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides etc.
  • a synthetic polymer preferably a cross-linked synthetic polymer, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides etc.
  • a commercially available product such as SourceTM (Amersham Biosciences AB, Uppsala, Sweden) can be surface-modified according to the invention.
  • the base matrix is made from a highly hydrophobic material, such as divinylbenzene and/or styrene.
  • ligands are immobilised to a particulate base matrix to a substitution level of about 10-200, such as about 20-100, e.g. about 40, ⁇ mol/ml base matrix to provide an RPC medium.
  • the reactive groups of the base matrix are allyl groups i.e. carbon- carbon double bonds.
  • the thiol group is coupled to allyl groups of the base matrix.
  • a commercially available base matrix which already exhibits allyl groups is used.
  • the allyl groups are provided according to well known methods.
  • the method also comprises a step of allylation of a base matrix to provide reactive groups.
  • the method can also comprise a step of activat- ing the reactive groups of the base matrix, e.g. by bromination.
  • the thiol group of the ligand is coupled to the base matrix via the allyl group of allyl glycidyl ether (AGE).
  • AGE allyl group of allyl glycidyl ether
  • steps (a) and (b) have been performed earlier to provide a ready-derivatised scaffold.
  • the present invention also encompasses a method, wherein the derivatisation of the scaffold has been performed earlier i.e. separate from the aminolysis and immobilisation to a base matrix of step (c) and (d), respectively.
  • the present invention is the use of homocysteine thiolactone in the preparation of a separation medium for hydrophobic interaction chromatography comprising a plurality of multimodal ligands. Details suitable for this aspect are e.g. as dis- closed above in the context of the method.
  • the invention is a kit for preparing a separation medium for hydrophobic interaction chromatography comprising a plurality of multimodal ligands, which kit comprises homocysteine thiolactone, one or more derivatisation agents and one or more amines in separate compartments as well as written instructions for its use.
  • the present kit comprises already derivatised homocysteine lactone and one or more amines in separate compartments as well as written instructions for its use.
  • the present invention is a ligand, which is multimodal in the sense that it comprises at least one hydrophobic group and at least one further group, which both allows for interaction with a single target molecule in hydrophobic interaction chromatography (HIC). Details regarding the interacting groups are e.g. as described above in the context of the present method. In a specific embodiment, the present ligand has been prepared using the method according to the invention and described above.
  • HIC hydrophobic interaction chromatography
  • the present invention relates to a separation medium comprising a plurality of ligands immobilised to a base matrix, wherein each ligand comprises at least one hydrophobic group and at least one further group, which both allows for interaction with a single target molecule in hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • the separation medium according to the invention is suitable for use either in conven- tional hydrophobic interaction chromatography (HIC) or in the specific kind of hydro- phobic interaction chromatography known as reverse phase chromatography (RPC), as discussed above.
  • a great advantage of the present separation medium is that it comprises more than one functionality that can interact with a target, which is a novel feature within the field of HIC. By providing such multiple functionalities, an improved adsorp- tion can be obtained, and its nature can be selected as desired depending on the choice of first and second interaction(s).
  • the present method allows an expedient preparation of multimodal HIC media, which to our knowledge has not been proposed before. Furthermore, the generality of the method provides easy access to the generation of libraries of multimodal HIC media. These libraries allow the possibility to screen for specific chromatographic characteristic. The present invention also encompasses such a library.
  • the present invention also encompasses a chromatography column packed with a medium according to the invention.
  • the column can be of any material and size, such as for large-scale production or lab-scale, or suitable for analytical purpose.
  • the column according to the invention is provided with luer adaptors, tubing connectors, and domed nuts.
  • a kit comprising a' chromatography column; a separation medium comprising the herein described multimodal ligands; and optionally liquids; in separate compartments together with instructions that describes use of the kit for isolation of biomolecules.
  • the invention also relates to a process of separating a target substance from a liquid, which process comprises to provide a separation medium which comprises a plu- rality of ligands immobilised to a base matrix, wherein each ligand comprises at least one hydrophobic group and at least one further group, which both allows for interaction with a single target molecule in hydrophobic interaction chromatography (HIC), and to contact said medium with the liquid and to allow adsorption of the target substance to the ligands.
  • the process also comprises a step of eluting the target substance from the separation medium by adding a liquid that desorbs the target compound from the separation medium.
  • the target substances are e.g.
  • biomolecules such as proteins, peptides, antibodies, virus, nucleic acids, such as DNA or RNA, plas- mids etc.
  • chromatography such as HIC and RPC, for separating a target substance as discussed above are well-known in this field, and the skilled person in this field can easily adopt the necessary parameters for use of the present process.
  • Figure 1 provides by Scheme 1 an illustration of how a diverse library of multimodal HIC/RPC media can be prepared according to the invention and using homocysteine thiolactone as a scaffold.
  • the multimodal HIC ligand is efficiently prepared in two syn- thetic steps.
  • the HIC interaction is herein first introduced by benzoylation of the scaffold.
  • the extra interactions can then conveniently be introduced by the opening of the ring structure be any suitable amine(s).
  • the multimodal HIC ligands so obtained will all contain a thiol group, which can subsequently be used to immobilise the ligand to an ac- tivated base matrix under conventional conditions.
  • Figure 2 shows a comparative elution profile of four proteins (myoglobine (1), ribonu- clease A (2), alpha-lactalbumine (3), and alpha-chymotrypsine (4)) on the prior art separation medium Phenyl SepharoseTM 6FF (Amersham Biosciences, Uppsala, Sweden), as described below.
  • Figure 3 shows the elution profile of four proteins (as defined under Figure 2) on Gel 2 according to the invention, as described below.
  • Figure 4 shows the elution profile of four proteins (as defined under Figure 2) on Gel 4b according to the invention, as described below.
  • Figure 5 shows the elution profile of four proteins (as defined under Figure 2) on Gel 7 according to the invention, as described below.
  • Figure 6 shows the elution profile of four proteins (as defined under Figure 2) on Gel 9 according to the invention, as described below.
  • Figure 7 shows a comparative elution profile of four proteins (as defined under Figure 2) on the prior art separation medium Phenyl SepharoseTM HP (Amersham Biosciences, Uppsala, Sweden), as described below.
  • Figure 8 shows the elution profile of four proteins (as defined under Figure 2) on Gel 11 according to the invention, as described below.
  • figures 2 to 8 show an illustrative comparison of the elution profiles of four proteins (ribonuclease, myoglobine, alpha-chymotrypsine and alpha- lactalbumine) using prior art separation media (high sub Phenyl SepharoseTM 6 Fast Flow and Phenyl SepharoseTM High Performance, Amersham Biosciences, Uppsala, Sweden) (above) and a multimodal HIC medium prepared according to the present invention (below).
  • prior art separation media high sub Phenyl SepharoseTM 6 Fast Flow and Phenyl SepharoseTM High Performance, Amersham Biosciences, Uppsala, Sweden
  • the elution profile obtained with the conventional phenyl derived HIC media show only two peaks, while some of the multimodal ligands prepared according to the present invention from the thiolactone scaffold, the peaks corresponding to each one of the four proteins are clearly separated.
  • Figure 9 shows the difference of elution profiles obtained with different multimodal media, namely Gels 1,2,4,5,8,10.
  • the primary hydrophobic interac- tion was first introduced by an aromatic substituent (see also Scheme 1) and the other interactions were then obtained by reacting this intermediate in a parallel fashion respectively with diethylene glycolamine (Gel 10), furfurylamine (Gel 8), 3-(Aminoethyl) pyridine (Gel 5), thiophene-2-methylamine (Gel 4a), butylamine (Gel 2a), benzylamine (Gel 1).
  • Deoxycortycosterone was applied on all columns under identical conditions and elution was performed in all cases with a linear gradient of increasing amount of organic solvent. The differences observed in the elution of deoxycortycosterone clearly emphasise the influence of the second interactions in RPC mode.
  • a solution A of DL-homocysteine thiolactone la and di-isopropylamine (DIPEA) in di- chloromethane (DCM) was cooled down to 0°C.
  • a solution B containing an acyl chloride or a sulfonyl chloride or an anhydride or an activated acid in DCM was cooled down to 0°C, and added drop wise to the solution A maintained between 0 and 5°C. The mixture was stirred overnight at room temperature. The solvent was removed under vacuum. If necessary the resulting product can be dissolved in ethyl acetate and washed with cit- ric acid (10% in water w/w) solution and by potassium carbonate (10% in water w/w) solution. The organic phase was washed with water before being dried with sodium sulphate and the solvent evaporated.
  • Step 2 The resulting product was dissolved in tetrahydrofuran (THF) and degassed by bubbling nitrogen for 10 min. To this solution an amine dissolved in THF was added at room temperature. The reaction mixture was stirred for another 17 hours. After the solvent has been evaporated under vacuum, ethyl acetate and citric acid (10% in water w/w) solution were mixed. The organic phase was washed with water before being dried with sodium sulphate and the solvent evaporated.
  • THF tetrahydrofuran
  • Brominated SepharoseTM 6 Fast Flow obtained following a well-known procedure was mixed with an alkaline solution of the product issued from step 2 and warmed up to 50 °C overnight. After reaction, the gel (1 volume) was filtered and washed with water (2x15 vol.), ethanol (2x15 vol.), acetic acid 0,2M (2x15 vol.) and water (2x15 vol.). The loading of ligand on the gel was measured by elementary analysis of Sulphur.
  • Step 1 11.5 g (75.0 mmol) of DL-homocysteine thiolactone hydrochloride was dissolved in 120 ml of DCM and 16.1 ml (150.0 mmol) of DIPEA. To this solution, 8.7 ml (75.0 mmol) of benzoyl chloride in 30 ml DCM were added slowly at room temperature according to Step 1. The solvent was removed under vacuum and ethyl acetate (300 ml) was added. The organic phase was washed with citric acid (10% in water w/w) solution and by potassium carbonate (10% in water w/w) solution (2x100 ml). The organic phase was dried with sodium sulphate before evaporation and 13.8 g of a white powder recovered. Yield: 83%.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.533 ml (4.9 mmol) of benzylamine were added and the mixture stirred at room temperature for 16h. The solvent removed, ethyl acetate (10 ml) was added to the crude material. Citric acid (10% in water w/w) solution (2x5 ml) was used to extract the excess of amine. The organic phase was dried over sodium sulphate before the solvent was removed under vacuum. A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50%) were also added to the ligand solution.
  • Step 1 same procedure as described in Example 1- Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.482 ml (4.9 mmol) of n- butylamine were added and the mixture stirred at room temperature for 16h. The solvent removed, ethyl acetate (10 ml) was added to the crude material. Citric acid (10% in water w/w) solution (2x5 ml) was used to extract the excess of amine. The organic phase was dried over sodium sulphate before the solvent was removed under vacuum. A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50% were also added to the ligand solution.
  • Step 1 same procedure as described in Example 1 -Step 1.
  • Step 2 82.3 mg (0.37 mmol) of product issued from Step 1 was dissolved in 5 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.184 ml (1.86 mmol) of n-butylamine were added and the mixture stirred at room temperature for 16h. The solvent removed, ethyl acetate (10 ml) was added to the crude material. Citric acid (10% in water w/w) solution (2x5 ml) was used to extract the excess of amine. The organic phase was dried over sodium sulphate before the solvent was removed under vacuum. A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50% were also added to the ligand solution.
  • Step 1 same procedure as described in Example 1- Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50% were also added to the ligand solution.
  • Step 1 same procedure as described in Example 1 -Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.500 ml (4.9 mmol) of 2- aminomethylthiophene were added and the mixture stirred at room temperature for 16h. The solvent removed, ethyl acetate (10 ml) was added to the crude material. Citric acid (10%) in water w/w) solution (2x5 ml) was used to extract the excess of amine. The organic phase was dried over sodium sulphate before the solvent was removed under vacuum. A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50%) were also added to the ligand solution. 6 ml of allylated SepharoseTM 6 Fast Flow with a loading of 65 ⁇ mol/ml of gel were activated using bromine and added to the ligand. The reaction mixture was shaken overnight at 50 °C. The gel 4a was then washed with THF, dimethylformamide (DMF), etha- nol, water, acetic acid 0.2M. Elementary analysis on dried gel: %S: 4.4 (calc. ligand concentration: 57.0 ⁇ mol/ml).
  • Step 1 same procedure as described in Example 1 -Step 1.
  • Step 2 82.3 mg (0.37 mmol) of product issued from Step 1 was dissolved in 5 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.191 ml (1.86 mmol) of 2-aminomethylthiophene were added and the mixture stirred at room temperature for 16h. The solvent removed, ethyl acetate (10 ml) was added to the crude material. Citric acid (10% in water w/w) solution (2x5 ml) was used to extract the excess of amine. The organic phase was dried over sodium sulphate before the solvent was removed under vacuum. A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of
  • Step 1 same procedure as described in Example 1- Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50% were also added to the ligand solution. 6 ml of allylated SepharoseTM 6 Fast Flow with a loading of 65 ⁇ mol/ml of gel were activated using bromine and added to the ligand. The reaction mixture was shaken overnight at 50 °C. The gel 5 was then washed with THF, dimethylformamide (DMF), etha- nol, water, acetic acid 0.2M. Elementary analysis on dried gel: %S: 2.1 (calc. ligand concentration: 61.6 ⁇ mol/ml).
  • Step 1 same procedure as described in Example 1 -Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.541 ml (4.9 mmol) of 1- methylpiperazine were added and the mixture stirred at room temperature for 16h. The solvent removed, and the crude material was purified by reversed phase HPLC (Wa- ter/Acetonitrile). A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50%) were also added to the ligand solution.
  • Step 1 same procedure as described in Example 1 -Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.591 g (4.9 mmol) of tris(hydroxymethyl)aminomethane hydrochloride and 3.76 ml of DIPEA were added and the mixture stirred at room temperature for 16h. The solvent removed, and the crude ma- terial was purified by reversed phase HPLC (Water/Acetonitrile). A colourless oil solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50% were also added to the ligand solution. 6 ml of allylated SepharoseTM 6 Fast Flow with a loading of 65 ⁇ mol/ml of gel were activated using bromine and added to the ligand. The reaction mixture was shaken overnight at 50 °C. The gel 7 was then washed with THF, dimethylformamide (DMF), etha- nol, water, acetic acid 0.2M. Elementary analysis on dried gel: %S: 1.30 (calc. ligand concentration: 21.9 ⁇ mol/ml). Example 8:
  • Step 1 same procedure as described in Example 1- Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.431 ml (4.9 mmol) of furfurylamine were added and the mixture stirred at room temperature for 16h. The solvent removed, and the crude material was purified by reversed phase HPLC (Wa- ter/Acetonitrile). A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50% were also added to the ligand solution. 6 ml of allylated SepharoseTM 6 Fast Flow with a loading of 65 ⁇ mol/ml of gel were activated using bromine and added to the ligand. The reaction mixture was shaken overnight at 50 °C. The gel 8 was then washed with THF, dimethylformamide (DMF), etha- nol, water, acetic acid 0.2M. Elementary analysis on dried gel: %S: 2.65 (calc. ligand concentration: 55.3 ⁇ mol/ml).
  • Step 1 same procedure as described in Example 1 -Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.817 g (4.9 mmol) of GLY-GLY amide hydrochloride and 4.76 ml of DIPEA were added and the mixture stirred at room temperature for 16h. The solvent removed, and the crude material was purified by reversed phase HPLC (Water/Acetonitrile). A white solid was recovered and the purity estimated by LC-MS (>90%) .
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of NaOH 50% were also added to the ligand solution.
  • Step 1 same procedure as described in Example 1- Step 1.
  • Step 2 220 mg (0.98 mmol) of product issued from Step 1 was dissolved in 7 ml of THF. To this degassed solution (10 min bubbling of nitrogen), 0.489 ml (4.9 mmol) of 2- (2aminoethoxy)ethanol were added and the mixture stirred at room temperature for 16h. The solvent removed, and the crude material was purified by reversed phase HPLC (Water/Acetonitrile). A white solid was recovered and the purity estimated by LC-MS (>90%).
  • Step 3 product from Step 2 was dissolved in 5 ml THF. 1 ml of water and 2ml of
  • Step 1 10.0 g (65.0 mmol) of DL-homocysteine thiolactone hydrochloride was dissolved in 120 ml of DCM and 23.9 ml (136 mmol) of DIPEA. To this solution, 7.7 ml (65.0 mmol) of valeryl chloride in 30 ml DCM were added slowly at room temperature according to Step 1. The solvent was removed under vacuum and ethyl acetate (400 ml) was added. The organic phase was washed with citric acid (10% in water w/w) solution and by potassium carbonate (10% in water w/w) solution (2x100 ml). The organic phase was dried with sodium sulphate before evaporation and 8.86 g of a white powder recovered. Yield: 68%.
  • Step 2 2.0 g (9.9 mmol) of product issued from Step 1 was dissolved in 30 ml of dried DMF. To this degassed solution (10 min bubbling of nitrogen), 3.0 g (24.8 mmol) of tris(hydroxymethyl)aminometha ⁇ e were added and the mixture stirred at 90°C for 4h. The solvent removed, a flash chromatography was realised starting with EtOAc/MeOH: 9/1 yielding 2.7 g of a white solid. Yield: 84 %.
  • Step 3 78.2 mg of product from Step 2 were dissolved in 2.5 ml of water and 2.5 ml of NaOH 50%) and the solution stirred for lh at room temperature. 6 ml of allylated SepharoseTM High Performance with a loading of 115 ⁇ mol/ml of gel were activated using bromine and added to the ligand solution. The reaction mixture was shaken overnight at 50 °C. The gel 11 was then washed successively with water , etha- nol, acetic acid 0.2M and water.
  • Test 1 Deoxycorticosterone
  • Injection volume lO ⁇ l (5mM Deoxycorticosterone in methanol)
  • Eluent A 10 mM Ammonium phosphate in MQ Milli-Q water, pH 7.
  • Detection wavelength 205, 236 and 280 nm
  • the chromatographic evaluation was done by separating a mixture of 4 angiotensin derivatives at four different pH values, pH 2,3, 7 and 12.
  • Injection volume lO ⁇ l (0.125 mg/ml) of each peptide in the sample mixture prepared in Milli-Q water)
  • Flow rate _1.0 ml/min.
  • the column volume in this case is 2.49 ml (the method was developed for ST4.5/150 steel columns)
  • the chromatograms of gels 2b, 4b, 7 and 9 made with a SepharoseTM 6 Fast Flow base matrix are presented in figures 3, 4, 5 and 6 respectively.
  • the four gels work under the tested HIC conditions described above and present different elution profiles.
  • the chromatogram of gel 11 made with a SepharoseTM High Performance base matrix is presented in figure 8. The gel works under the tested HIC conditions described above.
  • 1 Myoglobin 2: Ribonuclease A 3: ⁇ -lactalbumin 4: ⁇ -Chymotrypsin

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Pyridine Compounds (AREA)
  • Heterocyclic Compounds Containing Sulfur Atoms (AREA)
  • Furan Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé de préparation de ligands multimodaux destinés à la chromatographie d'interaction hydrophobe (CIH) qui consiste à utiliser un squelette cyclique comprenant un thiol, une amine et un groupe carbonyle; à former un dérivé de l'azote avec un réactif en vue de produire une interaction primaire; et à pratiquer l'aminolyse du dérivé résultant, ceci permettant d'introduire une interaction secondaire suite au carbonyle; de sorte qu'au moins une des interactions primaire et secondaire comprenne un groupe hydrophobe et qu'aucune desdites interactions ne comprenne de groupes chargés, c'est-à-dire de ligands échangeurs d'ions. Cette invention se rapporte également à un milieu de séparation comprenant de tels ligands multimodaux immobilisés sur une matrice de base.
PCT/SE2004/000316 2003-03-05 2004-03-05 Procede de preparation de ligands pour la chromatographie d'interaction hydrophobe Ceased WO2004078311A1 (fr)

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JP2006507945A JP4570052B2 (ja) 2003-03-05 2004-03-05 疎水性相互作用クロマトグラフィー用リガンドの製造法
CA2517757A CA2517757C (fr) 2003-03-05 2004-03-05 Procede de preparation de ligands pour la chromatographie d'interaction hydrophobe
EP04717913A EP1599265A1 (fr) 2003-03-05 2004-03-05 Procede de preparation de ligands pour la chromatographie d'interaction hydrophobe
US10/547,570 US7320755B2 (en) 2003-03-05 2004-03-05 Method of preparing ligands for hydrophobic interaction chromatography

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WO2007137752A1 (fr) * 2006-05-26 2007-12-06 Ge Healthcare Bio-Sciences Ab Procédé pour la génération de ligands présentant une affinité avec des agents chélatants métallifères
EP2684951A1 (fr) 2012-07-13 2014-01-15 Sandoz Ag Procédé de production d'une protéine recombinante d'intérêt
CN107513117A (zh) * 2017-08-24 2017-12-26 中国科学技术大学 一种基于硫内酯化学构筑的多功能非病毒基因输送载体聚合物
WO2025190679A1 (fr) * 2024-03-13 2025-09-18 Cytiva Bioprocess R&D Ab Ligand de chromatographie et matériau de chromatographie, et leurs utilisations

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JP4629660B2 (ja) * 2003-03-05 2011-02-09 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ マルチモードアニオン交換リガンドの製造法
SE0300612D0 (sv) * 2003-03-05 2003-03-05 Amersham Biosciences Ab A method of preparing ligands for hydrophobic interaction chromatography
JP5420531B2 (ja) * 2007-05-25 2014-02-19 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング カチオン交換クロマトグラフィーのためのグラフトコポリマー
ES2532408T3 (es) * 2009-10-30 2015-03-26 Xellia Pharmaceuticals Aps Procedimiento para purificar colistina y componentes de colistina purificados
JP2016535669A (ja) * 2013-10-10 2016-11-17 ジーイー・ヘルスケア・バイオプロセス・アールアンドディ・アクチボラグ クロマトグラフィー材料の製造方法
IL264631B2 (en) * 2016-08-16 2024-05-01 Regeneron Pharma A method for quantifying individual antibodies from a mixture

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007137752A1 (fr) * 2006-05-26 2007-12-06 Ge Healthcare Bio-Sciences Ab Procédé pour la génération de ligands présentant une affinité avec des agents chélatants métallifères
US7988858B2 (en) 2006-05-26 2011-08-02 Ge Healthcare Bio-Sciences Ab Method for generating metal chelating affinity ligands
EP2684951A1 (fr) 2012-07-13 2014-01-15 Sandoz Ag Procédé de production d'une protéine recombinante d'intérêt
WO2014009515A1 (fr) 2012-07-13 2014-01-16 Sandoz Ag Méthode de production d'une protéine recombinée présentant un intérêt
CN107513117A (zh) * 2017-08-24 2017-12-26 中国科学技术大学 一种基于硫内酯化学构筑的多功能非病毒基因输送载体聚合物
CN107513117B (zh) * 2017-08-24 2019-08-27 中国科学技术大学 一种基于硫内酯化学构筑的多功能非病毒基因输送载体聚合物
WO2025190679A1 (fr) * 2024-03-13 2025-09-18 Cytiva Bioprocess R&D Ab Ligand de chromatographie et matériau de chromatographie, et leurs utilisations

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US20060175258A1 (en) 2006-08-10
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